1. Field of the Invention
This invention relates to a method for determining HIV susceptibility to protease inhibitors.
2. Background of the Related Art
Treatment of the Human Immunodeficiency Virus type I (HIV-1) infection by antiretrovirals can lead to the selection of virus variants with decreased susceptibility to these agents. Near complete inhibition of HIV replication in vivo by triple combinations of reverse transcriptase inhibitors (RTIs) and of protease inhibitors (PIs), now often termed highly active antiretroviral therapy (HAART), is believed to significantly prevent emergence of resistance. However, in many patients treated by such drug combinations, HIV resistance to both RTIs and PIs can gradually develop, which appears to be mostly the case for patients in which the antiviral combination has failed to achieve a complete block of viral replication. In such patients, optimally active alternative treatment regimens need to be found, which will require careful measurement of HIV susceptibility to antiretroviral drugs. In addition, since antiviral combination therapy needs to be optimized to ensure the best long-term antiviral response, it could be essential to monitor HIV susceptibility profiles to antiretrovirals before the onset of therapy.
The monitoring of HIV resistance to antiretrovirals can be performed by genotypic analysis of the protease (PR) and of the reverse transcriptase (RT) coding regions of viral genomes carried in the plasma of infected patients. Nevertheless, the number of mutations in PR or in RT that are able to affect HIV susceptibility to a growing number of antiviral molecules is increasing considerably. Hence, the observed combinations of mutations, which are certainly an important marker of the evolution of resistance in the course of the treatment, cannot give precise indications on the actual level of susceptibility or resistance of the studied virus. Instead, only phenotypic assays, which directly measure inhibition of virus replication by antivirals in culture, can provide a quantitative assessment of resistance.
Several of the currently used phenotypic resistance assay systems examine the susceptibility of virus isolates obtained by co-culture of patient blood cells with donor primary peripheral blood mononuclear cells (PBMCs). These methods require several rounds of virus growth in primary donor cells for virus amplification, titration, and subsequent testing in the presence of the drugs. As a consequence they are costly and highly time-consuming.
More recently, an innovative technique, the recombinant virus assay (RVA), based on previous observations showing that deletions in transfected retroviral genomes could be repaired by homologous recombination, was proposed by Kellam and Larder. In this system, PCR-amplified viral RT sequences from patient plasma were cotransfected with a RT-deleted infectious molecular clone of HIV yielding a recombinant virus carrying the RT sequences of patient plasma virus. Since the molecular clone used for recombination was initially obtained from a laboratory-adapted HIV-1 strain, the recombinant virus could be conveniently tested on established cell lines instead of primary cell cultures.
Several versions of the RVA have been developed for HIV-1 susceptibility testing, which have yielded EC50 values that correlate well with corresponding HIV-1 RT or PR genotypes. However, in these recombinant systems, as well as in the PBMC assay, production of a testable stock of infectious particles requires amplification and titration of virus produced by exponential growth in lymphocytic cells, a step that requires cumulative rounds of viral replication and promotes genetic drift of the virus. Since several mutations able to confer resistance to RTIs or PIs have been shown to reduce the replicative capacity of the virus and to be selected against during drug-free HIV replication, there exists a need in the art for a susceptibility assay for HIV that would only require one step of virus replication.
This invention aids in fulfilling this need in the art by providing an in vitro, single cycle, recombinant virus assay (RVA) for determining inhibition of HIV replication by a protease inhibitor. The method comprises transfecting a human epithelial cell line with: (1) amplified HIV protease sequences of an HIV virus to be assayed for resistance to the protease inhibitor; (2) an HIV, envelope defective, molecular clone for recombination, wherein the molecular clone has a complete deletion of its protease coding sequence and an envelope deletion so that no HIV envelope is expressed by the HIV molecular clone; and (3) a plasmid containing VSV-G envelope coding sequence under the control of a promoter for phenotypic complementation of the HIV, envelope defective, molecular clone. The resulting transfected cells are contacted with the protease inhibitor and cultured to repair the protease coding sequence by homologous recombination between the HIV molecular clone and the amplified HIV protease sequences, and to produce a testable stock of infectious particles by coexpression of the protease-repaired HIV molecular clone and the VSV-G envelope coding sequence, such as a VSV-G envelope coding sequence. Indicator cells containing an indicator gene are contacted with the testable stock of infectious particles without amplification of the infectious particles prior to infecting the indicator cells. The indicator gene is expressed in the indicator cells, and accumulation of indicator gene product is determined as a measure of HIV replication. The susceptibility of the recombinant virus to protease inhibitors is determined by contacting transfected cells with serial dilutions of the inhibitor, allowing to calculate the concentration of inhibitor able to inhibit 50 or 90% of virus infectivity [IC50 and IC90].
This invention provides a single-cycle version of RVA for testing HIV resistance to protease inhibitors. Similar to the previously described RVA systems, the assay of the invention uses recombination of PCR-amplified HIV sequences from plasma with a deleted HIV molecular clone. In one embodiment of the invention, cotransfection can be performed in human epithelial cells (e.g., HeLa cells) that are subsequently treated with serial dilutions of protease inhibitors, since the inhibitory action of these compounds is only perceptible upon treatment of virus-producing cells, yielding a small stock of testable virus particles. The resulting particle stocks can be subsequently tested for infectivity on a HeLa-derived P4 cell line, which allows precise and quantitative titration of HIV on the basis of a single round of replication.
To increase the sensitivity of the test and to make sure that replication of the tested particles in the P4 cells is limited to a single cycle, in a preferred embodiment of the invention, the HIV envelope coding sequences can be removed from the HIV plasmid clone, and phenotypic complementation of the envelope ensured by coexpression of an exogenous viral envelope protein, such as VSV-G envelope glycoprotein.
Overall, the results obtained with the assay of the invention match the genotypes of the tested viruses and the treatment history of the corresponding patients. Since the assay of the invention is particularly rapid (5 days on average, not including the PCR), the assay is useful for routine monitoring of HIV drug susceptibility in treated HIV-infected patients.